Process for the preparation and purification of the sodium salt of hyaluronic acid

ABSTRACT

The present invention concerns a process for the preparation and purification of the sodium salt of HA from the fermentation broth of Streptococcus or Bacillus or from rooster combs, the sodium salt of HA obtained and purified with said process and pharmaceutical, cosmetic and nutritional compositions comprising said sodium salt of HA.

The present invention concerns a process for the preparation andpurification of the sodium salt of hyaluronic acid.

FIELD OF THE INVENTION

Hyaluronic acid (HA) is a high-molecular-weight linear, anionicpolysaccharide, free of sulfate groups, consisting of alternatingresidues of D-glucuronic acid and N-acetyl-D-glucosamine.

It is present in nature in pericellular gels, in the fundamentalsubstance of the connective tissue of vertebrate organisms (of which itrepresents one of the main components), in the synovial fluid ofarticulations, in the vitreous humor and in the umbilical cord.

HA therefore plays an important role in the biological organism, aboveall as a mechanical support of cells of numerous tissues, such as skin,tendons, muscles and cartilage.

It is also known that HA, through its membrane receptors, in particularCD44, CD54 and CD168, modulates many different processes relating to thephysiology and biology of cells, such as, for example, proliferation,migration, cell differentiation and angiogenesis, and which also exertsother functions such as the hydration of tissues and lubrication of thearticulations. It is absolutely biocompatible and, thanks to itsnumerous special features, is widely used in various fields, from tissuerepair to viscous additional therapy, from dermo-aesthetic medicine tothe intraocular surgery, from tissue engineering to cell therapy, andmuch more.

The physico-chemical and biological features of HA are stronglycorrelated to its molecular weight (MW) which is extremely variable: itcan generally be asserted that the weight average MW of HA ranges from20,000 to 13×10⁶ Da approximately, and this approximation is necessaryas it changes radically in relation to the source and production andpurification method used for isolating it.

There are fundamentally two main methods for obtaining HA:

production from animal source: historically HA is extracted from animaltissues such as the umbilical cord, vitreous humour or bovine synovialliquid and, above all, rooster combs. Its production from animal sourcehas many limitations, it is expensive, for example, as numerous passagesare necessary for eliminating various types of impurities (starting fromthe mass of organic residues after the digestion of the startingtissue), it therefore requires passages which ensure the inactivationand elimination of any contaminant agent (such as viruses) possiblypresent in the starting material, it requires the availability ofconsiderable quantities of raw material and does not produce greatyields;

fermentation of microorganisms: some microorganisms, in particular ofthe genus Streptococcus or Pasteurella, suitably stimulated and/ormodified, are capable of producing HA which is secreted in the culturebroth from which it is isolated through various processes known toskilled persons in the field. Also in this case, numerous passages arenecessary for eliminating the “impurities” present such as, for example,the residues of the cell walls of the microorganisms used, metal ions,nucleic acids and any other undesired proteinaceous material. In spiteof these limitations, this is, to date, the most developed and widelyused production method of HA. New methods are being studied for theproduction of HA via bio-technology, through the transfection of genesexpressing the enzyme HA-synthases in suitable host cells, such as somekinds of Bacillus (Megaterium and Sibtilis) and in Escherichia coli. Allprocedures suitable for eliminating any potentially harmful residue arehowever also necessary for these production methods.

In any case, regardless of the method used, a key passage in theproduction of HA is obviously the extraction and purification phase ofthe polysaccharide. The known methods are numerous and extremelyarticulated, obviously modulated with respect to the starting sourcesfor obtaining HA. First of all, the residues of the source must beeliminated, consequently, for extraction from animal tissue there aredigestion phases of the proteins, and subsequent filtrations,centrifugations and washings; for the fermentation, centrifugations andprogressive washings are normally used. In any case, a liquid fractionis obtained, from which the polysaccharide is isolated. In this respect,the best known and certainly most widely-applied procedure, above allfor HA from animal sources, is precipitation with solvents: in short,increasing concentrations of organic solvents (ethanol, acetone) areused on the liquid fraction mentioned above, which cause theprecipitation of the hyaluronic acid, which is then purified by means ofsubsequent solubilizations and precipitations.

An alternative system envisages the use of quaternary salts,cetylpyridinium or cetyltrimethylammonium with the aim of complexing thepolysaccharide and causing its precipitation. Subsequent solubilizationsand precipitations are again necessary before obtaining the finalproduct.

The development of techniques has also combined the key steps describedabove, so as to make the process efficient in terms of yield andeffective in terms of purity: to date, however, there are still numerousadverse events, in the order of a few hundreds, reported to thecompetent authorities (such as FDA) that have arisen especially afteradministration of injectable pharmaceutical compositions based on HA.

This polysaccharide is used in a wide variety of fields and pathologies:from cosmetic applications (by topical or oral administration) with amoisturizing action, to topical dermocosmetic use having a soothingeffect, from injecting devices for the correction of skin defects(intradermal) whether they be wrinkles or scars, up to more strictlypharmacological applications such as intra-articular use in bone andjoint diseases, or intraocular use as a substitute for the vitreoushumour, and so forth.

Whereas for cosmetic applications, which do not involve damaged tissues,a cosmetic-grade HA (less pure) is sufficient, it is evident however,that in the case of injectable pharmaceutical applications (especiallyin closed cavities such as articulation and the eye), a degree ofabsolute purity is necessary: the presence of various types ofcontaminants, such as nucleic acids and/or proteins and/or residualbacterial toxins of the cell walls of Gram-positives such aslipoteichoic acid LTA (for example of the genus Bacillus, Streptococcus,Enterococcus and Staphylococcus) or of Gram-negatives such aslipopolysaccharide LPS (such as, for example, Escherichia Coli,Pasteurella and Salmonella), can cause a significant inflammatoryreaction with the consequent release of cytokines (in particular TNF andIL-1) at both a local and systemic level, which could trigger ageneralized inflammatory reaction with repercussions in the wholeorganism, reaching (in the most serious cases) forms of septic shock,and this explains the numerous reports of adverse events cited above.LTA and LPS, in fact, are polymers consisting of a lipid portion and asaccharide portion capable of causing strong immune responses and, inthe most serious cases, arthritis, nephritis, meningitis or causingfever and shock with consequences that can even be fatal.

It should also be taken into account that, as previously mentioned, theMW of HA varies in relation to the source and production method. Morespecifically, the MW indicated to herein, refers to the weight averagemolecular weight measured with the “intrinsic viscosity” method. Thevariability of the MW determines the use of HA in different fields: forexample, low MWs are applied in dermatological or dermocosmeticpreparations (about 200 kDA; Connectivine®), whereas for intra-articularapplications, higher molecular weights are preferred (normally withinthe range of 700-1800 kDA; Hyalgan®, Hyalubrix® Orthovisc®) up to MWs ofover 1500 kDA used in plastic surgery or intraocular applications. It isextremely important to perfectly calibrate the MW of HA, not onlybecause it determines the biological and physico-chemicalcharacteristics of the polymer, but also because it has been amplydemonstrated that HA having a MW lower than 30,000 Da has a stronginflammatory effect (EPO 138572), which is absolutely undesirable,regardless of the application.

This means that in the production and purification process of HA,various factors must be evaluated and controlled:

-   -   the process yield: it is fundamental to extract the maximum        possible amount of HA from the production source selected;    -   the accuracy of the ensemble of the purification steps: the        product obtained must be free of any contaminant capable of        triggering inflammatory processes;    -   the fractionation of the MW: the desired MW and certainty of        having eliminated the inflammatory fractions must be obtained.

Numerous attempts at combining these requirements are known in the stateof the art. Among these the following can be schematically mentioned:

U.S. Pat. No. 5,925,626: purification of HA from rooster combs byprecipitation with ethanol and formation of two MW fractions (50-100 kDaand 500-730 kDa), free of the inflammatory fraction;

EP535200: purification of HA from rooster combs by salification withquaternary amines and subsequent solvent precipitation (ethanol oracetone). A HA is obtained having a MW ranging from 750 to 1230 kDa,free of inflammatory fractions and specifically destined for ophthalmicuse;

U.S. Pat. No. 6,489,467: purification of HA from Streptococcus by forcedacidification using HCl, with subsequent variations in the pH anddiafiltrations obtaining HA having a MW of about 1700 kDa;

Choi et al. Biomaterials Research, 2014, 18, 1-10: purification of HAfrom Streptococcus zooepidemicus by diafiltration and precipitation withacetone. A HA with a MW ranging from 900 to 1100 kDa is obtained;

EP2870255: purification of HA from Streptococcus zooepidemicus byfiltrations and ultrafiltrations, pH variations reaching a MW rangingfrom 60 to 2400 kDa, and final precipitation with ethanol.

DETAILED DESCRIPTION OF THE INVENTION

An object of the present invention relates to a new process for thepreparation and purification of a sodium salt of HA which allows itsproduction with

-   -   a very high purity degree as it is free of contaminants and    -   a precise and specific MW.

The new purification process of hyaluronic acid and its sodium saltcomprises various steps, articulated and sequential, for thepurification of a HA prepared as widely known to skilled persons in thefield: from both a biological source, in particular from bird crests ofthe genus Gallus (EP0138572, hereinafter these crests will be indicatedas rooster combs, regardless of the gender of the bird) and also fromthe fermentation process of Streptococco, also applicable to a HAprepared with molecular engineering techniques from Bacillus Subtilisand Bacillus Megaterium (EP 2614088, EP2614087); this process ispreferably applicable to a HA obtained from the fermentation broth ofStreptococcus, even more preferably from the broth of Streptococcus equisub-sp. equi, 68222, mutant H-1 (EP0716688). The Applicant hasdemonstrated hereunder how this process allows the preparation not onlyof a sodium salt of HA in conformance with all the physico-chemicalspecifications required by the European Pharmacopoeia (monography of HA:Ph, Eur, 5.0, 01/2005: 1472) but, in particular, for some puritycharacteristics, the specifications claimed by the Applicant have beenfurther restricted, in order to guarantee a hyaluronic acid having avery high degree of purity, which can be used in complete safetyespecially in all injectable pharmaceutical compositions(intra-articular, intradermal and intraocular) as it is free of anypro-inflammatory and pyrogenic component. The sodium salt of HA purifiedby means of the new process object of the invention, can also be used inthe preparation of all the derivatives known to skilled persons in thefield, such as, for example, its salts with heavy metals (EP 0827514),esters (EP 0216453), amides (EP 1095064) sulfonates (EP0940410) andcrosslinked products, among which self-crosslinked products (EP0341745).

An object of the invention also relates to particular thermal treatmentphases of HA still to be purified, in particular the fermentation brothof Streptococcus (containing this HA), in order to obtain differentfractions of HA having a precise weight average MW: the Applicant has infact perfected specific thermal treatment in terms of temperature andtreatment time (conditions described in detail hereunder) which followthe inactivation phase of the fermentation broth or which take placecontemporaneously with the enzymatic digestion of the rooster combs,which allow a final product having the desired intrinsic viscosity, tobe obtained. Specific weight average MW values of HA correspond tospecific values of the intrinsic viscosity, and this viscosity iscalculated according to what is written in the corresponding monographyof HA of the European Pharmacopoeia according to the “intrinsicviscosity” method (Ph. Eur. 5.0, 01/2005: 1472).

A further object of the invention relates to pharmaceutical, cosmeticand nutritional compositions containing said fractions, morespecifically:

-   -   pharmaceutical compositions for intra-articular use containing        sodium salt of HA with a very high/high/medium weight average MW        to be used in the visco-supplementation of arthritic joints, in        traumatic joint damage, in subchondral damage;    -   pharmaceutical compositions for intraocular use or for ocular        administration for the treatment of eye diseases, containing        sodium salt of HA with a very high/high/medium/low weight        average MW;    -   pharmaceutical compositions containing sodium salt of HA with a        very high/high/medium/low weight average MW to be used in the        prevention of post-surgical adhesions;    -   pharmaceutical compositions for topical or injective use        (intradermal and/or intramuscular) containing sodium salt of HA        with a very high/high/medium/low weight average MW, preferably        sodium salt of HA with a medium/low weight average MW, in the        treatment of skin ulcers, bedsores, burns, scars and skin        lesions, in the treatment of keloids or hypo/hypertrophic scars,        therefore in the treatment of all types of skin defects with        intact or damaged skin, and as a therapy for the treatment of        skin diseases such as eczemas and various kinds of dermatitis,        in particular atopic dermatitis and nappy rash, psoriasis;    -   pharmaceutical compositions for intravesical use containing        sodium salt of HA with a very high/high/medium weight average        MW, in particular for the treatment of interstitial cystitis;    -   pharmaceutical compositions for injective use containing sodium        salt of HA with a very high/high/medium weight average MW as a        filler in the dermo-aesthetic field or as body shaping in        plastic surgery;    -   cosmetic compositions for topical and oral use;    -   pharmaceutical or nutritional compositions containing sodium        salt of HA with a very high/high/medium/low weight average MW        for the oral treatment or arthritic joints, for tendon trophism,        skin trophism and of the gastrointestinal mucous membrane.

A further object of the invention also relates to two/three-dimensionalbiomaterials comprising derivatives prepared with HA purified accordingto the invention, in the form of pads, woven, non-woven fabrics,granulates, films and gels, also in possible association with cells ofvarious origins and/or blood components, such as, for example,platelet-derivatives. The Applicant has perfected the followingpurification process with the main objective of eliminating all theimpurities deriving from the selected production source of hyaluronicacid, mainly represented by proteins, nucleic acids and/or byother/various kinds of pyrogens. In particular, the objective of thepresent invention is the complete elimination of bacterial toxinsderiving from Gram-positive bacteria such as Streptococcus or Bacillus(or from bacteria such as, for example, Enterococci and Staphylococci)or from Gram-negative bacteria such as, for example Escherichia Coli (orPasteurella and Salmonella) normally absent in the fermentation broths(if not residual from the source strain) but whose possiblecontamination would represent a very important safety problem: thepresence of toxins (such as LTA or LPS) in HA as end-product would infact deprive it of the necessary purity required, as it can determinethe production of highly pro-inflammatory factors that could causeinflammation/infection of the joints or tissues treated, even up totheir total destruction or necrosis.

The new purification process of the sodium salt of HA ensures:

-   -   a high process yield    -   total purity of the product    -   production of the fraction with the desired intrinsic viscosity        (therefore MW).

The process consists of two or three steps:

-   -   inactivation (this step is present only for the production of HA        from the fermentation broth of Bacillus and of Strepococcus);    -   extraction;    -   purification.

Inactivation: this step relates to the production of HA fromStreptococcus (and also from Bacillus) which is fermented in specificfermenters containing suitable culture mediums under the conditionsknown to skilled persons in the field; this process is followed by theinactivation phase of the bacteria by acidification of the culturebroth, preferably with HCl, in order to decrease the pH to a valuebetween 4 and 5, at this value in fact Streptococcus completely ceasesits metabolic activity.

This is followed by the thermal treatment of the inactive broth byheating (described hereunder) for the production of a HA having a highor medium viscosity or with a low viscosity; this treatment is noteffected in the case of the production of HA having a very highviscosity. As indicated above, in fact, specific MW ranges correspond tospecific viscosity ranges and in this way the Applicant has developed aprocess which allows the production of the desired fractions withabsolute precision, as demonstrated hereunder.

The biomass is eliminated by filtering the inactivated broth on pads ofCelite (diatomaceous earth, chemical name: silica dioxide), with apossible further subsequent filtration through a filter with afiltration degree equal to 0.5 μm (propylene filters are preferable);the broth is preferably neutralized with soda (NaOH) at a pH of 6.5-7.5.

Extraction: this phase is in common for both the production of HA fromStreptococcus and Bacillus and for HA obtained from rooster combs; inthe latter case, the process according to the invention starts from thehomogenate obtained from combs according to what is described in EP0138572. More specifically, the homogenate, is subjected to thermaltreatment by heating (described hereunder) for the production of a HAhaving a high or medium or low viscosity; this thermal treatment iseffected contemporaneously with the enzymatic digestion (to which saidhomogenate—hereinafter defined as enzymatic digested homogenate—issubjected) with the enzyme papain prepared in a phosphate buffer, asknown to skilled persons in the field. The non-purified hyaluronic acidpresent in the neutralized filtrate or present in the mixture ofenzymatic digest subjected to thermal treatment (hereinafter defined asmedium containing non-purified HA), is subsequently complexed with CPC(Cetyl Pyridinium Chloride, the CPC-HA salt is formed) after a furtheraddition of Celite under stirring. The complex is left to settle toseparate the solid from the liquid phase which is eliminated. The HApresent in the solid phase is then solubilized under stirring, with aNaCl saline solution and the product obtained (sodium salt of HA solublein this medium), is subjected to further filtrations/purifications bymeans of filtering cloths to separate the residual Celite and by meansof filters with a filtration degree equal to 3 μm (polypropylene filtersare preferred), collecting the filtrate. This particular procedure isdefined as “extraction” of the sodium salt of HA not yet purified, andcan be effected from 1 to 4 times. After collecting and joining thefiltered products, the so “extracted” product is treated with particularresins of the aromatic type suitable for absorbing large-sized moleculesthanks to the pore radius ranging from 200 to 300 Angstrom, theydecisively contribute to lowering the total impurity of HA deriving fromthe system of origin from which the polysaccharide was purified and alsofrom the substances used in the above process. The resin consisting ofcrosslinked polystyrene matrixes is preferably used, the resin DIAIONHP20 (or HP20L) (MITSUBISHI CHEMICAL) has proved to be particularlyefficient. Said treatment consists of leaving resin and extract understirring for a period of time. The product obtained is then filtered bymeans of filters/cloths preferably made of polypropylene (to separatethe resins from the HA sodium salt), possibly also on 3 μm-filtrationdegree filters.

Purification: this final step can be possibly preceded by theprecipitation in ethanol of the sodium salt of HA obtained in theextraction” phase (this step can be introduced in order to furtherpurify the polysaccharide, especially when it comes from rooster combs);the elimination of the above solvent is followed by there-solubilization of the precipitate in water, subsequently proceedingwith the following “purification” steps: addition of NaOH in water forthe total elimination of the residual toxins, neutralization, preferablywith HCl (at 37% by weight), up to a pH ranging from 8 to 9 (the term“neutralization” is simply used in this phase for indicating theApplicant's intention of lowering the pH to values closer toneutrality), filtration preferably by means of filters having afiltration degree of 3 μm, precipitation and at least a washing withethanol, final washing in an organic solvent, preferably acetone. Thesodium salt of HA thus produced and purified is dried as known toskilled persons in the field.

An object of the present invention therefore relates to the preparationand purification process of the sodium salt of HA schematized hereunderin its phases:

A. Inactivation (for the purification of a HA produced from thefermentation of Streptococcus and Bacillus):

-   -   A1. acidification of the fermentation broth to a pH of 4-5; HCl        1N is preferably used;    -   A2. thermal treatment of the broth, under stirring (this        treatment is not effected if a HA with a very high viscosity is        produced);    -   A3. elimination of the biomass by means of filtration on pads of        Celite (chemical name: silica dioxide; in an amount of from 20        to 60 g/litre of broth, preferably 30-40 g/litre), possible        further filtration with filters having a filtration degree of        0.5 μm, preferably polypropylene filters;    -   A4. neutralization to pH 6.5-7.5, preferably with aqueous NaOH        at 20%.

B. Extraction:

in the case of homogenate from combs, the corresponding thermaltreatment is first effected contemporaneously with its enzymaticdigestion and subsequent filtration (to eliminate the undigestedbiological residue), followed by the following common phases:

-   -   B1. addition of Celite (in an amount of from 20 to 60 g/litre of        broth/litre of enzymatic digest, i.e. per litre of medium        containing non-purified HA) and complexing with Cetyl Pyridinium        Chloride (CPC) (4-20 g/litre/litre of enzymatic digest,        preferably 5-15 g/litre), under stirring, for at least 30        minutes and subsequent sedimentation for at least 30 minutes;    -   B2. elimination of the liquid phase;    -   B3. solubilization of the HA present in the solid phase in NaCl        (an 0.3M aqueous solution is preferably used) under stirring for        a period of 4 to 24 h, filtration by means of filtering cloths        to separate the residual Celite and filters with a filtration        degree of 3 μm (polypropylene filters are preferred) and        collection of the first extract as sodium salt of HA; this        procedure should be repeated from 1 to 4 times;    -   B4. joining the extracts;    -   B5. addition to the joined extracts of a resin of the aromatic        type (in an amount of from 10 to 60 g/litre of extract) with a        pore radius of 200-300 Angstrom, the resin composed of        crosslinked polystyrene matrixes is preferred, the resin DIAION        HP20 (or HP20L) is even more preferred, this treatment is        effected under stirring for at least 8 h;    -   B6. at least a filtration by means of filtering cloths        (preferably made of polypropylene) to separate the resins from        the sodium salt of HA, and possibly at least a filtration with 3        μm-filtration degree filters (for this filtration, polypropylene        filters are preferred).

C. Purification:

in the case of the sodium salt of HA obtained from rooster combs, thisstep can be possibly preceded by the precipitation in ethanol of thesodium salt of HA obtained in the previous step, with the elimination ofthe above solvent and ri-solubilization of the precipitate in purifiedwater (Ph. Eur. 8.0, 01/2009:0008) to restore the starting volume andsubsequently proceeding with the following purification phases,regardless of the source selected:

-   -   C1. addition of NaOH (a 0.2-0.4 M solution is preferably used)        in water, under stirring;    -   C2. neutralization to a pH ranging from 8 to 9, preferably with        HCl (at 37%);    -   C3. filtration, a filter with a filtration degree of 3 μm is        preferable (polypropylene filters are preferred);    -   C4. precipitation and at least a washing of the sodium salt of        HA sodium salt coming from step C3 with ethanol, final washing        in an organic solvent, preferably acetone;    -   C5. drying of the sodium salt of HA as known to skilled persons        in the field, preferably from 25 to 40° C. for not less than        15h, under vacuum.

Determination of the Weight Average MW of the Sodium Salt of HA

The thermal treatment object of the present invention allows theproduction of the sodium salt of HA with an intrinsic viscosity (IV)which falls within specific ranges (IV measured according to the methoddescribed in Ph. Eur. 5.0. 01/2005; Ph. Eur. 1472), described hereunder:

Thermal Treatment of HA from Fermentation Broth of Streptococcus orBacillus:

60±5° C. for 5-40 minutes: this allows the production of a HA with ahigh viscosity, therefore a sodium salt of HA having a final IV withinthe range of 17-24 dl/g; the above thermal treatment is preferablycarried out at 65° C. for 5-30 minutes;

70±5° C. for 5-40 minutes: this allows the production of a HA with amedium viscosity, therefore a sodium salt of HA having a final IV withinthe range of 10-15 dl/g; the above thermal treatment is preferablycarried out at 70° C. for 5-30 minutes;

90±5° C. for 150-300 minutes: this allows the production of a HA with alow viscosity, therefore a sodium salt of HA having a final IV withinthe range of 3-6 dl/g;

If the thermal treatment is not effected, the final IV of the sodiumsalt of HA, purified according to the object of invention, is equal toor over 29 dl/g, therefore the purified product is a sodium salt of HAhaving a very high viscosity.

Thermal Treatment of HA from Rooster Combs:

50-60° C. for 26-30 h: this allows the production of a HA with a highviscosity, therefore a sodium salt of HA having a final IV within therange of 17-24 dl/g; the above thermal treatment is preferably carriedout at 55° C. for 28 h;

60-65° C. for 28-30 h: this allows the production of a HA with a mediumviscosity, therefore a sodium salt of HA having a final IV within therange of 10-15 dl/g; the above thermal treatment is preferably carriedout at 60° C. for 30 h;

65-70° C. for 46-50 h: this allows the production of a HA with a lowviscosity, therefore a sodium salt of HA having a final IV within therange of 3-6 dl/g; the above thermal treatment is preferably carried outat 65° C. for 48 h.

At the end of the treatment, a skilled person in the field can collect asample and verify the viscosity obtained, on the basis of the resultreached, he can either repeat the operation or modify the time and/ortemperature of the treatment (still within the range described) in orderto reach the desired viscosity: the treatment times and temperatures forreaching the ranges of IV described above depend, in fact, on theconcentration and MW of the HA present in the initial broth/digest.

The Mark-Houwink equation (Terbojevich M. et al, Carbohydrate Research,1986, 149, 363-377; Terbojevich M. et al, Carbohydrate Research, 1986,157, 269-272) is used for specifying the corresponding average MWs, theequation relates the VI with the MW. Consequently, the viscosity rangescorrespond to specific MW ranges:

29 dl/g corresponds to about 1885 kDa

17-24 dl/g corresponds to a range from about 920 to 1450 kDa

10-15 dl/g corresponds to a range from about 450 to 780 kDa

3-6 dl/g corresponds to a range from about 90 to 231 kDa.

With the following experimentation, the Applicant has demonstrated theefficacy of the process object of the invention, in terms of purity ofthe obtained sodium salt of HA

Purification of the Sodium Salt of HA Produced from Streptococcus

At the end of the fermentation process of Streptococcus equi 68222mutant H-1, fermented as in Example 3 of EP 0716688, about 5 litres ofbroth are collected and contaminated with E. coli ATCC 8739 in aquantity of 10⁹ bacterial cells per ml of broth. This broth is then leftin an open container (in order to favour further contamination frombacteria or from yeast/fungi whose spores may be present in the air) fora time not shorter than 16 h, at room temperature. It is then dividedinto two samples: 2.5 litres (sample A) are subjected to the completepurification process object of the invention, whereas the remaining 2-5litres (sample B) are subjected to simple precipitation, as describedhereunder. A sample of broth is subjected to microbiological control byqualitative and quantitative analyses (by means of the API Systemeffected according to Ph. Eur. 5.0; 2.6.12) of the microbial and/ormycotic charge present. The results are indicated in table A.

Sample A: the broth is subjected to inactivation by acidification to pH4.3 with HCl 1 N and thermal treatment at 65° C. for 10 minutes, understirring. The biomass is eliminated by filtration on Celite (40 g/l) andneutralized to pH 6.5 with aqueous NaOH at 20%. This is followed by theextraction phase with the addition of Celite (20 g/l of broth) andcomplexing with CPC (10 g/l of broth) under stirring for 30 minutes withsubsequent sedimentation for 1 h; the liquid phase is then eliminated bysiphoning and the HA present in the solid phase is solubilized inaqueous NaCl 0.3 M, still under stirring for a period of 20 hours; thisprocess continues with filtration on a filtering cloth and also usingfilters with a filtration degree of 3 μm and the collection of the firstextract; this procedure is repeated 2 times and the two extracts arejoined; this is followed by the addition of a resin of the aromatictype, DIAION HP20 (40 g/l, this treatment is effected, under stirring,for 8 h) and filtration to separate the resins from the HA usingpolypropylene filtering cloths. The purification is carried out byadding NaOH 0.4 M in water (under stirring) and is followed byneutralization at pH 8.5 with HCl (37%) and by filtration with filtersin polypropylene with a filtering degree of 3 μm. The sodium salt of HAis precipitated with ethanol 100%, washed with ethanol 80%, and thefinal washing is effected in acetone; the final drying of the sodiumsalt of HA obtained and purified, is effected at 25° C. for 20 h undervacuum.

Sample B: this sample was subjected to inactivation by acidification atpH 4.3 with HCl 1 N and thermal treatment at 65° C. for 10 minutes understirring, exactly as for Sample A. The biomass was eliminated from thebroth by filtration on Celite, the filtrate was then subjected toprecipitation with ethanol with drying, at 25° C., of the HA (notpurified) precipitated, for 20 h under vacuum.

The purified HA must be non-pyrogenic, i.e. there should be no elementsthat can cause an increase in the body temperature after itsadministration. The test for evaluating the non-pyrogenicity can becarried out in various ways: the LAL Test (for the specificdetermination in vitro of the endotoxin LPS deriving fromGram-negatives, required by Ph. Eur. 5.0 in the monography relating toSodium Hyaluronate), the Pyrogen Test (non-discriminating analysis invitro on the nature of the pyrogen agent) and Endosafe®-IPT (test invitro not required by Pharmacopoeia).

LAL Test: the basis of the LAL test is the capacity of the amebocytesextracted from blood of Limulus polyphemus to gel in the presence ofbacterial endotoxins from Gram-negatives, primarily responsible for thepyrogen effect. This test is carried out according to Ph. Eur. 5.0,2.6.14.

Pyrogen Test: this test represents the most widely-used analysis methodfor determining the presence of pyrogen substances, it contemplates theuse of rabbits into which a small dose of the product is injected in theouter ear vein, the baseline temperature is taken three hours after theinjection. The rise in temperature is a sign of pyrogenicity of theproduct. This test is carried out according to Ph. Eur. 5.0, 2.6.8.

Endosafe®-IPT (Charles River Laboratories, Inc.): test capable ofdetecting the presence of pyrogens of any type as they can stimulate theproduction of cytokine IL-1β which is highly pro-inflammatory. This testallows the identification of pyrogens of both an endotoxic (LPS) ornon-endotoxic nature (LTA and/or proteins and/or pyrogen derivatives,for example from viruses or yeast/mold): it consists of two steps, inthe first step, the sample is incubated with human blood (if present,pyrogens stimulate the production of IL-1β by the monocytes of theblood), the second step consists of the detection of the presence ofIL-1β produced by a specific test ELISA read at 450 nm (Schindler S. etal, ALTEX, 2009, 26, 265-277).

For samples A and B the analysis of the pyrogens was carried out usingthe Pyrogen Test and Endosafe®-IPT, as the LAL Test is not capable ofevaluating the possible presence of pyrogen agents other than endotoxinLPS, whereas the other two methods reveal pyrogens of any nature, evenif with different sensitivities. The IPT test, in fact, allows bacterialresidues coming from both Gram-positives and Gram-negatives to beidentified, and surpasses the Pyrogen Test as far as the sensitivity isconcerned. Furthermore, the IPT is more specific than the test onrabbits as it evaluates the toxicity of the contaminants in human tissue(Hartung T. et al, ATLA, 2001, 29, 99-123). For both samples, the totalprotein content determined as from Ph. Eur. 5.0, 01/2005; 1472, was alsoevaluated. The results of the tests are shown in table B.

Results

TABLE A in this table, the presence can be observed of an importantbacterial and also mycotic charge in terms of non-pathogen organisms andpathogen organisms such as B. Cereus, Coli and Candida. Microbial chargeBacillus Cereus 4.6 × 10⁷/ml Streptococcus 2.2 × 10⁷/ml E. Coli 9.8 ×10⁶/ml Total charge 7.8 × 10⁷/ml Mycotic charge Mold 9.4 × 10⁷/ml Yeast2.5 × 10⁷/ml (Candida Lusitaniae) Total charge 1.2 × 10⁸/ml

The table therefore demonstrates how the initial broth specificallypolluted contained pyrogen elements from both Gram-positives andGram-negatives, in addition to various kinds of pyrogens, such asderivatives of Candida

TABLE B Pyrogen Test IPT Protein content Sample A 0.9° C. N.R. 0.04%Sample B 4.1° C. >5 EU/mg  10% NR: non-detectable (lower than 0.05EEU/mg)

These data show that the new purification process is capable ofpurifying HA from various kinds of pyrogen agents:

It can be noted from Table B that the IPT test of sample B shows a veryhigh value of pyrogen toxins, the monography of HA Ph. Eur. 5.0,01/2005: 1472, in fact, allows, for injective administrations of HA, amaximum limit of endotoxins lower than 0.05 IU/mg (i.e. 0.05 EU/mg). Insample B, there is therefore a pyrogen concentration at least 100 timeshigher than this limit. In the three rabbits treated for the Pyrogentest, this concentration causes an overall temperature increase of 4.1°C. According to Ph. Eur. 5.0, 2.6.8, the product satisfies the Pyrogentest, if the sum of the three temperature rises does not exceed thevalue of 1.15°, therefore sample B proved to be strongly pyrogenic, thusconfirming the data of the IPT test. Finally, this sample has a veryhigh total protein % deriving from the mediums used for the fermentationof Streptococcus, and from the same bacteria not completely eliminated:monography of HA Ph. Eur. 5.0, 01/2005: 1472 limits, by parenteraladministrations, the overall proteins to a maximum value not exceeding0.1%, therefore, also in this case, sample B has a protein value about100 times higher than the limit value.

Sample A, purified according to the object of the invention, satisfiesall the requirements for the injective administration of the sodium saltof HA: pyrogens in vivo with a temperature rise lower than 1.15° C.,toxins in vitro lower than 0.05 EU/mg, and overall protein content lowerthan 0.1% (Ph. Eur. 5.0, 01/2005: 1472).

This result demonstrates the effectiveness of the process object of theinvention, as it ensures the total purification of the sodium salt of HAeven from a particularly polluted sample, from both endotoxins andvarious kinds of pyrogens, guaranteeing the safety of the product which,in this way, satisfies all the requirements also in terms of morelimiting purities, as demonstrated hereunder.

EXAMPLE 1 Preparation and Purification of the Sodium Salt of HA fromStreptococcus with IV within the Range of 10-15 dl/g

At the end of the fermentation process of Streptococcus equi (68222mutant H-1) fermented as per Example 3 of EP0716688), 5 litres of brothare inactivated by acidification to pH 4.5 with HCl 1N. This is followedby the thermal treatment of the broth with a temperature increase to 70°C. for 20 minutes under stirring. The broth is then filtered and pouredinto a Buchner filter in which 200 g of Celite were prepared on afiltering cloth. At the end of the filtration, the product isneutralized with aqueous NaOH 20% and the pH is fixed at 7.0. 100 g ofdiatomaceous earth and subsequently CPC in an amount equal to 11 g/l ofbroth, are added, under stirring, for 30 minutes, to the filtered broth.The whole mixture is left to rest for 40 minutes to allow thesedimentation of the newly formed CPC-HA complex. The liquid phase iseliminated by siphoning. The HA present in the solid phase issolubilized by means of a solution of aqueous NaCl 0.3M, under stirringfor 10 h. The sodium salt of HA is finally filtered through a filteringcloth and filtering cartridges having a filtration degree of 3 μm(Pall). 200 g of Diaion HP20 resins are added to the extract which isleft under stirring for 10 h. The whole mixture is filtered on propylenecloth and then, in sequence, through filters (Pall) with a 3 μmfiltration degree. Aqueous NaOH is added to the solution of extracts,which is neutralized with HCl (at 37%), bringing the pH to a value of8.5. The extracts are then filtered through a 3 μm-filtration degreefilter. The solution of sodium salt of HA is precipitated with ethanoland kept under stirring for 30 minutes. The product is left to settlefor 10 minutes and the supernatant is eliminated by siphoning. Theproduct is washed with ethanol (under stirring for 10 minutes), and thesupernatant is then eliminated by siphoning (in alternative, in case ofbroth quantities higher than 5 liters, the solid product is recovered byfiltration on a filtering cloth). The last washing with acetone iscarried out and the solid is recovered by filtration on a filteringcloth. The product obtained is positioned on suitable stainless steeltrays and dried for 22 h at a temperature of 25° C. under vacuum.

Analysis of the product obtained (according to Ph. Eur. 5.0, 01/2005:1472):

IV: 14.5 dl/g (weight average MW: 748,000 Daltons)

Proteins: 0.02%

Bacterial endotoxins (LAL test): 0.012 EU/mg

Yield: for the determination of the total yield of the process object ofthe invention, the concentration is determined of HA in carbazole (Ph.Eur. 5.0, 01/2005: 1472) present in the broth at the end of thefermentation, and is related to the final concentration of HA obtainedat the end of the purification process (i.e. the ratio is calculatedbetween the quantity in grams of end-product vs litres of broth at theend of the fermentation (then subjected to purification), a simpleproportion is subsequently calculated to obtain the yield valueexpressed as percentage of purified HA vs initial HA to be purified).

In this case, 3.3 g/litre of HA were determined in the broth at the endof the fermentation, and 3.0 g/l of HA as purified product. The finalyield was therefore higher than 90%.

EXAMPLE 2 Preparation and Purification of the Sodium Salt of HA fromRooster Combs with IV within the Range of 10-15 dl/g

250 g of dry powder prepared from rooster combs as described in Example1 of EP0138572, are mixed with 0.29 g of papain in 10 litres of dibasicsodium phosphate/dihydrate sodium phosphate/EDTA buffer (pH 6.5), understirring for 10 minutes. This mixture is then subjected to thermaltreatment by increasing its temperature to 60° C. for 30 hours. Theresulting homogenate is then filtered and discharged into a Buchner inwhich 200 g of Celite have been prepared in a polypropylene filteringcloth. 200 g of Celite are added to the filtered product under stirringand then 2 litres of aqueous CPC solution (at 29 g/l) are added to saidfiltered product, leaving under stirring for 30 minutes. The mixture isthen left to rest for 40 minutes to allow the sedimentation of the newlyformed CPC-HA complex and the liquid phase is eliminated by siphoning.The HA present in the solid phase is solubilized with a solution of 4litres of aqueous NaCl 0.3 M, under stirring for 10 hours. Finally, thesodium salt of HA is filtered through a filtering cloth and filteringcartridges having a filtration degree of 3 μm (Pall). At this point, 200g of Diaion HP20L resins are added to the extract and the mixture isleft under stirring for 10 hours. The whole mixture then is filtered onpolypropylene cloth and subsequently, in sequence, through 3μm-filtration degree filters (Pall). The sodium salt of HA isprecipitated with 1.8 volumes of ethanol, under stirring for 30 minutes;the product is left to settle and the supernatant is eliminated bysiphoning. The sedimented product is re-solubilized with 5 litres ofpurified water, under stirring.

Aqueous NaOH 0.2 M is added to the solution obtained, which isneutralized with HCl (37%), bringing the pH to 8.2. The filtration iscontinued using 3 μm-filtration degree filters. The sodium salt of HAobtained is then precipitated with ethanol under stirring for 30minutes, the product is left to settle for 10 minutes and thesupernatant is eliminated by siphoning. The product is washed withethanol, the product is left to settle for 10 minutes and thesupernatant is the eliminated by siphoning (in alternative, in case ofbroth quantities higher than 5 liters, the solid product is recovered byfiltration on a filtering cloth). The last washing with acetone iscarried out and the solid is recovered by filtration on a filteringcloth. The product obtained is positioned on suitable stainless steeltrays and dried for 22 hours at a temperature of 40° C. under vacuum.

Analysis of the product obtained (according to Ph. Eur. 5.0, 01/2005:1472):

IV: 14 dl/g (weight average MW: 714,000 Daltons)

Proteins: 0.04%

Bacterial endotoxins (LAL test): 0.012 EU/mg

Yield: in this case, for the determination of the total process yield,the concentration of HA in carbazole is determined per litre ofenzymatic digest, and is related to the final concentration of HAobtained at the end of the purification process (i.e. the ratio iscalculated between the quantity in grams of the end-product vs litres ofenzymatic digest (then subjected to purification), a simple proportionis then calculated to obtain the yield value expressed as percentage ofpurified HA vs initial HA to be purified).

In this case, the final yield of HA was higher than 85%.

Example 3 Preparation and Purification of the Sodium Salt of HA fromStreptococcus with IV within the Range of 3-6 dl/g

The procedure is the same as that described in Example 1, but thethermal treatment carried out is at 90° C. for 250 minutes. The finalproduct is dried for 25 hours at 40° C., under vacuum.

Analysis of the product obtained (according to Ph. Eur. 5.0, 01/2005:1472):

IV: 5 dl/g (weight average MW: 181,000 Daltons)

Proteins: 0.015%

Bacterial endotoxins (LAL test): 0.0075 EU/mg

Yield: In this case, 3.3 g/litre of HA were determined in the broth atthe end of the fermentation, and 2.5 g/litre as purified product. Thefinal yield was therefore higher than 75%.

For this reason, a further object of the present invention relates tothe purification process of HA wherein the maximum values of totalproteins and toxins claimed for the sodium salt of HA, in addition tothe total yield at the end of the process are:

−0.05% of proteins vs 0.1% established in Ph. Eur. 5.0, 01/2005: 1472;

−002 IU/mg of endotoxins vs the maximum limit of 0.05 IU/mg allowed inPh. Eur. 5.0, 01/2005: 1472;

Yield: from 75 to 90%.

1. A process for the preparation and purification of the sodium salt ofHA from the fermentation broth of Streptococcus or Bacillus or fromrooster combs comprising the following steps: B. extraction, comprisingthe following steps: B1. addition of Celite to a medium containingnon-purified HA and complexing of HA with Cetyl Pyridinium Chloride(CPC), under stirring for at least 30 minutes and followingsedimentation for at least further 30 minutes; B2. elimination of theliquid phase formed in step B1; B3. solubilization of the HA present inthe solid phase in an aqueous solution of NaCl and collection of a firstextract as sodium salt of HA; this procedure B3. being effected from 1to 4 times; B4. joining the extracts coming from step/s B3.; B5.addition of an aromatic resin having a pore radius ranging from 200 to300 Angstrom to the joined extracts of B4, the resin composed ofcrosslinked polystyrene matrixes being preferred, leaving under stirringfor at least 8 hours; B6. filtration of the mixture coming from step B5;C. purification, comprising the following steps C1. addition of anaqueous solution of NaOH to the filtrate obtained from step B6.; C2.neutralization to a pH ranging from 8 to 9, preferably with HCl; C3. atleast one filtration; C4. precipitation and at least one washing of thesodium salt of HA obtained in step C3. with ethanol, final washing in anorganic solvent, preferably acetone; C5. drying of the sodium salt ofHA, preferably between 25 and 40° for not less than 15 h, under vacuum.2. The process for the preparation and purification of the sodium saltof HA according to claim 1 wherein the fermentation broth ofStreptococcus is a fermentation broth of Streptococcus equi sub specieequi 68222, mutant H-1.
 3. The process for the preparation andpurification of the sodium salt of HA from the fermentation broth ofStreptococcus or Bacillus according to claim 1, wherein the processcomprises a previous initial step of inactivation A. of the fermentationbroth, comprising the following steps: A1. acidification of thefermentation broth to pH 4-5, preferably with HCl; A2. elimination ofthe biomass from Streptococcus or Bacillus by at least one filtration;A3. neutralization to pH 6.5-7.5, preferably by means of NaOH.
 4. Theprocess for the preparation and purification of the sodium salt of HAaccording to claim 3, wherein the deactivation phase comprises a thermaltreatment of the fermentation broth by heating for the production of ahigh- medium- or low-viscosity HA.
 5. The process for the preparationand purification of the sodium salt of HA according to claim 4, whereinthe thermal treatment is carried out by increasing the temperature ofthe broth to 60±5° C. for 5-40 minutes for the production of a sodiumsalt of HA having a final intrinsic viscosity (IV) within the range of17-24 dl/g, the thermal treatment being preferably carried out at 65° C.for 5-30 minutes.
 6. The process for the preparation and purification ofthe sodium salt of HA according to claim 4, wherein the thermaltreatment is carried out by increasing the temperature of the broth to70±5° C. for 5-40 minutes for the production of a sodium salt of HAhaving a final intrinsic viscosity (IV) within the range of 10-15 dl/g,the thermal treatment being preferably carried out at 70° C. for 5-30minutes.
 7. The process for the preparation and purification of thesodium salt of HA according to claim 4, wherein the thermal treatment iscarried out by increasing the temperature of the broth to 90±5° C. for150-300 minutes for the production of a sodium salt of HA having a finalintrinsic viscosity (IV) within the range of 3-6 dl/g.
 8. The processfor the preparation and purification of the sodium salt of HA fromrooster combs according to claim 1, wherein the extraction step ispreceded by a thermal treatment of a homogenate of rooster combs byheating and contemporary enzymatic digestion, for the production of ahigh-, medium- or low-viscosity HA.
 9. The process for the preparationand purification of a sodium salt of HA according to claim 8, whereinthe thermal treatment is effected by increasing the temperature to50-60° C. for 26-30 h to prepare a sodium salt of HA having a finalintrinsic viscosity (IV) within the range of 17-24 dl/g, the thermaltreatment being preferably carried out at 55° C. for 28 h.
 10. Theprocess for the preparation and purification of a sodium salt of HAaccording to claim 8, wherein the thermal treatment is effected byincreasing the temperature to 60-65° C. for 28-30 h to prepare a sodiumsalt of HA having a final intrinsic viscosity (IV) within the range of10-15 dl/g, the thermal treatment being preferably carried out at 60° C.for 30 h.
 11. The process for the preparation and purification of thesodium salt of HA according to claim 8, wherein the thermal treatment iseffected by increasing the temperature to 65-70 ° C. for 46-50 h, forthe production of a sodium salt of HA having a final intrinsic viscosity(IV) within the range of 3-6 dl/g, the thermal treatment beingpreferably carried out at 65° C. for 48 h.
 12. The process for thepreparation and purification of the sodium salt of HA from rooster combsaccording to claim 1, wherein the purification step C. is preceded by aprecipitation in ethanol of the sodium salt of HA obtained in theextraction step B., elimination of the solvent and solubilization of theprecipitate in purified water.
 13. A process for the preparation andpurification of the sodium salt of HA from the fermentation broth ofStreptococcus or Bacillus according to claim 1, comprising the followingsteps: A. inactivation of the fermentation broth, comprising thefollowing steps: A1. acidification of the broth to pH 4-5, HCl 1N beingpreferably used; A2. possible thermal treatment of the broth, understirring; A3. elimination of the biomass from Streptococcus or Bacillusby filtration on pads of Celite in an amount of from 20 to 60 g/litre ofbroth, preferably 30-40 g/l, possible filtration with filters having afiltration degree of 0.5 μm, polypropylene filters being preferred; A4.neutralization to pH 6.5-7.5, preferably with aqueous NaOH at 20%; B.extraction, comprising the following steps: B1. addition to the filtrateA3 of Celite in an amount of from 20 to 60 g/litre of broth andcomplexing with CPC 4-20 g/litre, preferably 5-15 g/litre, understirring for at least 30 minutes and subsequent sedimentation for atleast a further 30 minutes; B2. elimination of the liquid phase; B3.solubilization of the HA present in the solid phase in aqueous NaCl 0.3M under stirring for a period ranging from 4 to 24 h, filtration bymeans of filters having a filtration degree of 3 μm and collection ofthe first extract as sodium salt of HA; this procedure B3. beingeffected from 1 to 4 times; B4. joining the extracts; B5. addition of anaromatic resin in an amount of from 10 to 60 g/litre of extract, havinga pore radius ranging from 200 to 300 Angstrom, to the joined extractsof B4., the resin composed of crosslinked polystyrene matrixes beingpreferred, leaving under stirring for at least 8 h; B6. filtrationscomprising filtering cloths, possibly also filters with a filtrationdegree of 3 μm, polypropylene filters being preferred; C. purification,comprising the following steps C1. addition of NaOH 0.2-0.4 M in water,under stirring, to the filtered product B6.; C2. neutralization to a pHranging from 8 to 9, preferably with HCl; C3. filtration, a filtrationdegree equal to 3 μm being preferred, polypropylene filters being morepreferred; C4. precipitation and at least one washing of HA sodium saltobtained from step C3., with ethanol, final washing in an organicsolvent, preferably acetone; C5. drying of the sodium salt of HA asknown to skilled persons in the field, preferably between 25 and 40° fornot less than 15 h, under vacuum.
 14. The process for the preparationand purification of the sodium salt of HA from rooster combs accordingto claim 1, wherein the extraction step B. is preceded by thermaltreatment of a homogenate of combs by heating and contemporary enzymaticdigestion, for the production of a high- or medium- or low-viscosity HA,comprising the following steps: B. extraction, comprising the followingsteps: B1. addition to the enzymatic digested of Celite in an amount offrom 20 to 60 g/litre of enzymatic homogenate and complexing with CPC inan amount of from 4 to 20 g/litre, preferably 5-15 g/litre, understirring for at least 30 minutes and subsequent sedimentation for atleast a further 30 minutes; B2. elimination of the liquid phase; B3.solubilization of the HA present in the solid phase in aqueous NaCl 0.3M under stirring for a period ranging from 4 to 24 h, filtration bymeans of filters having a filtration degree of 3 μm and collection of afirst extract as sodium salt of HA; this procedure B3. being effectedfrom 1 to 4 times; B4. joining the extracts coming from step/s B3.; B5.addition of an aromatic resin in an amount of from 10 to 60 g/litre ofextract, having a pore radius ranging from 200 to 300 Angstrom, to thejoined extracts of B4., preferably a resin composed of crosslinkedpolystyrene matrixes, leaving under stirring for at least 8h; B6.filtrations comprising filtering cloths, possibly also filters with afiltration degree of 3 μm, polypropylene filters being preferred; C.Purification, comprising the following steps: addition of NaOH 0.2-0.4 Min water, under stirring, to the filtered product of B6.; C2.neutralization to a pH ranging from 8 to 9, preferably with HCl; C3.filtration, a filtration degree equal to 3 μm being preferred,preferably using polypropylene filters; C4. precipitation and at leastone washing of HA sodium salt obtained from step C3., with ethanol,final washing in an organic solvent, preferably acetone; C5. drying ofthe sodium salt of HA, preferably at a temperature between 25 and 40°for not less than 15 h, under vacuum.
 15. The process for thepreparation and purification of the sodium salt of HA from rooster combsaccording to claim 14, wherein the purification step C. is preceded by aprecipitation in ethanol of the sodium salt of HA obtained in theextraction step B., elimination of the solvent and solubilization of theprecipitate in purified water.
 16. The process for the preparation andpurification of the sodium salt of HA according to claim 1, wherein theyield of the process ranges of from 75 to 90% and the maximum values oftotal proteins and toxins of the sodium salt of the HA obtained are: a.0.05% of proteins; b. 0.02 IU/mg of endotoxins.
 17. A sodium salt of HAprepared and purified according to claim 1, having a finale intrinsicviscosity (IV) equal to or higher than 29 dl/g.
 18. A sodium salt of HAprepared and purified according to claim 5, having a finale intrinsicviscosity (IV) within the range of 17-24 dl/g.
 19. A sodium salt of HAprepared and purified according to claim 6, having a finale intrinsicviscosity (IV) within the range of 10-15 dl/g.
 20. A sodium salt of HAprepared and purified according to claim 7, having a finale intrinsicviscosity (IV) within the range of 3-6 dl/g.
 21. Pharmaceutical,cosmetic and nutritional compositions according to claim 17, for use a.in the treatment of arthritic joints, traumatic joint damage,subchondral damage; b. in the treatment of eye diseases; c. in thetreatment of post-surgical adhesions; d. in the treatment of skinulcers, bedsores, burns, scars and skin lesions, keloids orhypo/hypertrophic scars, all types of skin defects with intact ordamaged skin; e. in the treatment of skin diseases such as eczemas andvarious kinds of dermatitis, in particular atopic dermatitis and nappyrash, psoriasis; f. for the treatment of interstitial cystitis. 22.Pharmaceutical and cosmetic compositions according to claim 17, for usein the dermo-aesthetic field or as body shaping in plastic surgery. 23.Cosmetic and nutritional compositions according to claim 17, for topicaland oral use.
 24. Pharmaceutical and nutritional compositions accordingto claim 17, for use in the oral treatment or arthritic joints, fortendon trophism, skin trophism and of the gastrointestinal mucousmembrane.
 25. A sodium salt of HA prepared and purified according toclaim 17, for the preparation of HA derivatives, preferably its saltswith heavy metals, esters, amides, sulfonates and crosslinked productsamong which self-crosslinked products are preferred.
 26. A sodium saltof HA prepared and purified according to claim 17, for the preparationof two/three-dimensional biomaterials in the form of pads, woven,non-woven fabrics, granulates, films and gels, also in possibleassociation with cells of various origins and/or blood components, suchas, for example, platelet-derivatives.